Multi-Niche Human Bone Marrow On-A-Chip for Studying the Interactions of Adoptive CAR-T Cell Therapies with Multiple Myeloma.

Multiple myeloma (MM), a cancer of bone marrow plasma cells, is the second-most common hematological malignancy. However, despite immunotherapies like chimeric antigen receptor (CAR)-T cells, relapse is nearly universal. The bone marrow (BM) microenvironment influences how MM cells survive, proliferate, and resist treatment. Yet, it is unclear which BM niches give rise to MM pathophysiology. Here, we present a 3D microvascularized culture system, which models the endosteal and perivascular bone marrow niches, allowing us to study MM-stroma interactions in the BM niche and model responses to therapeutic CAR-T cells. We demonstrated the prolonged survival of cell line-based and patient-derived multiple myeloma cells within our in vitro system and successfully flowed in donor-matched CAR-T cells. We then measured T cell survival, differentiation, and cytotoxicity against MM cells using a variety of analysis techniques. Our MM-on-a-chip system could elucidate the role of the BM microenvironment in MM survival and therapeutic evasion and inform the rational design of next-generation therapeutics. TEASER: A multiple myeloma model can study why the disease is still challenging to treat despite options that work well in other cancers.

Understanding and improving cellular immunotherapies against cancer: From cell-manufacturing to tumor-immune models.

The tumor microenvironment (TME) is shaped by dynamic metabolic and immune interactions between precancerous and cancerous tumor cells and stromal cells like epithelial cells, fibroblasts, endothelial cells, and hematopoietically-derived immune cells. The metabolic states of the TME, including the hypoxic and acidic niches, influence the immunosuppressive phenotypes of the stromal and immune cells, which confers resistance to both host-mediated tumor killing and therapeutics. Numerous in vitro TME platforms for studying immunotherapies, including cell therapies, are being developed. However, we do not yet understand which immune and stromal components are most critical and how much model complexity is needed to answer specific questions. In addition, scalable sourcing and quality-control of appropriate TME cells for reproducibly manufacturing these platforms remain challenging. In this regard, lessons from the manufacturing of immunomodulatory cell therapies could provide helpful guidance. Although immune cell therapies have shown unprecedented results in hematological cancers and hold promise in solid tumors, their manufacture poses significant scale, cost, and quality control challenges. This review first provides an overview of the in vivo TME, discussing the most influential cell populations in the tumor-immune landscape. Next, we summarize current approaches for cell therapies against cancers and the relevant manufacturing platforms. We then evaluate current immune-tumor models of the TME and immunotherapies, highlighting the complexity, architecture, function, and cell sources. Finally, we present the technical and fundamental knowledge gaps in both cell manufacturing systems and immune-TME models that must be addressed to elucidate the interactions between endogenous tumor immunity and exogenous engineered immunity.

Keeping Myeloma in Check: The Past, Present and Future of Immunotherapy in Multiple Myeloma.

Multiple myeloma is an incurable disease of malignant plasma cells and an ideal target for modern immune therapy. The unique plasma cell biology maintained in multiple myeloma, coupled with its hematological nature and unique bone marrow microenvironment, provide an opportunity to design specifically targeted immunotherapies that selectively kill transformed cells with limited on-target off-tumor effects. Broadly defined, immune therapy is the utilization of the immune system and immune agents to treat a disease. In the context of multiple myeloma, immune therapy can be subdivided into four main categories: immune modulatory imide drugs, targeted antibodies, adoptive cell transfer therapies, and vaccines. In recent years, advances in all four of these categories have led to improved therapies with enhanced antitumor activity and specificity. In IMiDs, modified chemical structures have been developed that improve drug potency while reducing dose limiting side effects. Targeted antibody therapies have resulted from the development of new selectively expressed targets as well as the development of antibody drug conjugates and bispecific antibodies. Adoptive cell therapies, particularly CAR-T therapies, have been enhanced through improvements in the manufacturing process, as well as through the development of CAR constructs that enhance CAR-T activation and provide protection from a suppressive immune microenvironment. This review will first cover in-class breakthrough therapies for each of these categories, as well as therapies currently utilized in the clinic. Additionally, this review will explore up and coming therapeutics in the preclinical and clinical trial stage.

A multi-niche microvascularized human bone marrow (hBM) on-a-chip elucidates key roles of the endosteal niche in hBM physiology.

The human bone marrow (hBM) is a complex organ critical for hematopoietic and immune homeostasis, and where many cancers metastasize. Understanding the fundamental biology of the hBM in health and diseases remain difficult due to complexity of studying or manipulating the BM in humans. Accurate biomaterial-based in vitro models of the hBM microenvironment are critical to further our understanding of the BM-niche and advancing new clinical interventions. Here we report a unique, 96-well format, microfluidic hBM-on-a-chip that incorporates the endosteal, central marrow, and perivascular niches of the human BM. Osteogenic differentiation of donor human mesenchymal stromal cells (MSCs) produced robust mineralization on the bottom surface ("bone-like endosteal layer") of the device, and subsequent seeding of human endothelial cells and MSCs in a fibrin-collagen hydrogel network ("central marrow") on the top created an interconnected 3D microvascular network ("perivascular niche"). The 96-well format allows eight independent "chips" to be studied in one plate, thereby increasing throughput and reproducibility. We show that this complex, multi-niche microtissue accurately mimics hBM composition and microphysiology, while providing key insights on hematopoietic progenitor dynamics. Presence of the endosteal niche decreased the proliferation and increased maintenance of CD34+ hematopoietic stem cells (HSCs). Upon exposure to radiation, HSCs in the hBM-chips containing endosteal niches were less frequently apoptotic, suggesting a potentially radio-protective role of the osteoblast surface. Our methods and results provide a broad platform for creating complex, multi-niche, high-throughput microphysiological (MPS) systems. Specifically, this hBM-on-a-chip opens new opportunities in human bone marrow research and therapeutics development, and can be used to better understand normal and impaired hematopoiesis, and various hBM pathologies, including cancer and BM failures.

Sample-to-analysis platform for rapid intracellular mass spectrometry from small numbers of cells.

Real-time, advanced diagnostics of the biochemical state within cells remains a significant challenge for research and development, production, and application of cell-based therapies. The fundamental biochemical processes and mechanisms of action of such advanced therapies are still largely unknown, including the critical quality attributes that correlate to therapeutic function, performance, and potency and the critical process parameters that impact quality throughout cell therapy manufacturing. An integrated microfluidic platform has been developed for in-line analysis of a small number of cells via direct infusion nano-electrospray ionization mass spectrometry. Central to this platform is a microfabricated cell processing device that prepares cells from limited sample volumes removed directly from cell culture systems. The sample-to-analysis workflow overcomes the labor intensive, time-consuming, and destructive nature of existing mass spectrometry approaches for analysis of cells. By providing rapid, high-throughput analyses of the intracellular state, this platform enables untargeted discovery of critical quality attributes and their real-time, in-process monitoring.

Posttranscriptional m(6)A Editing of HIV-1 mRNAs Enhances Viral Gene Expression.

Covalent addition of a methyl group to adenosine N(6) (m(6)A) is an evolutionarily conserved and common RNA modification that is thought to modulate several aspects of RNA metabolism. While the presence of multiple m(6)A editing sites on diverse viral RNAs was reported starting almost 40 years ago, how m(6)A editing affects virus replication has remained unclear. Here, we used photo-crosslinking-assisted m(6)A sequencing techniques to precisely map several m(6)A editing sites on the HIV-1 genome and report that they cluster in the HIV-1 3' untranslated region (3' UTR). Viral 3' UTR m(6)A sites or analogous cellular m(6)A sites strongly enhanced mRNA expression in cis by recruiting the cellular YTHDF m(6)A "reader" proteins. Reducing YTHDF expression inhibited, while YTHDF overexpression enhanced, HIV-1 protein and RNA expression, and virus replication in CD4+ T cells. These data identify m(6)A editing and the resultant recruitment of YTHDF proteins as major positive regulators of HIV-1 mRNA expression.